DOI: 10.1111/eea.

12848
SPECIAL ISSUE: INSECTS IN AGROECOSYSTEMS

Intercropping impacts the host location behaviour and

population growth of aphids
Agathe Mansion-Vaqui
e1* , Aur
elie Ferrer1, Felipe Ramon-Portugal2, Alexander Wezel1
& Alexandra Magro2,3
1
Research Unit Agroecology and Environment, ISARA-Lyon, 23 rue Jean Baldassini, Lyon 07 69364, France, 2University of
Toulouse – ENSFEA, 2 rt de Narbonne, Castanet-Tolosan 31326, France, and 3UMR CNRS/UPS/IRD 5174 EDB
(Laboratoire Evolution et Diversite Biologique), Toulouse F-31062, France
Accepted: 6 May 2019

Key words: disruptive crop hypothesis, bottom-up pest control, Sitobion avenae, cereal, legume,
Hemiptera, Aphididae, soft winter wheat, winter pea, white clover

Abstract Increasing intrafield plant diversity has been shown to regulate pest populations in various agro-
ecosystems. Among the suggested mechanisms for this bottom-up pest control, the disruptive crop
hypothesis states that herbivores’ abilities to locate and colonize their host plants are reduced by the
presence of non-host plants. Under laboratory conditions, we evaluated how intercropping wheat
and legumes modifies the behaviour of apterous cereal aphids, Sitobion avenae (Fabricius) (Hemi-
ptera: Aphididae), in terms of host plant location and population growth. We compared two inter-
cropping systems – soft winter wheat, Triticum aestivum L. (Poaceae), associated with winter pea,
Pisum sativum L., or with white clover, Trifolium repens L. (both Fabaceae) – and sole stands of soft
winter wheat. Aphids needed more time to locate their wheat host plant and then spent less time on
wheat when it was intercropped with clover. At the population level, and accounting for host plant
biomass, only intercropping wheat with clover significantly reduced aphid densities on wheat, as this
was particularly disruptive to S. avenae behaviour and population growth. Our laboratory study
points out that the species used as non-host plants and their density are important parameters that
should be taken into account in field studies on intercropping systems.

simultaneous cultivation of two cash crops, and ‘compan-

Introduction
The link between biodiversity and ecosystem functioning
has long been accepted (Naeem et al., 2002) and many
experimental studies have shown that diversity is a key
determinant of ecosystem processes, such as plant produc-
tivity (Tilman, 2015). Currently, a paradigm change is
underway in agroecosystem management, with attempts
to increase biodiversity at different spatio-temporal scales,
from the crop field, to the farm, and finally across the land-
scape (Garibaldi et al., 2017). At the field level, increased
diversity is mainly achieved by intercropping (Andow,
1991), defined as ‘the cultivation of two or more species of
crop in such a way that they interact agronomically’ (Van-
dermeer, 1989). We may distinguish two types of inter-
cropping: ‘true intercropping’ (Willey, 1979), that is the
*Correspondence: Agathe Mansion-Vaqui
e, Research Unit Agroecol-
ogy and Environment, ISARA-Lyon, 23 rue Jean Baldassini, Lyon 07
69364, France. E-mail: agathe.mansion-vaquie@hotmail.fr
ion cropping’, the cultivation of a cash crop and a benefi-
cial non-crop (Ben-Issa et al., 2017; Verret et al., 2017). An
increase in productivity in intercropping systems may be
achieved through an improved yield per unit area, due to
complementary use of resources, facilitation, and/or
increased pest regulation (Brooker et al., 2015).
In this study, we are interested in the role of intercrop-
ping on the control of aphids (Hemiptera: Aphididae),
which are among the main pests in temperate regions
(Dedryver et al., 2010). We focus on cereals and legumes
because cereals are important in most temperate farming
systems and are dominant crops in terms of agricultural
area, and the cultivation of legumes for animal feed, food,
and other ecosystem services (e.g., supply of nitrogen) is
increasing (Stagnari et al., 2017). Intercropping cereals
and legumes has several agronomical and environmental
advantages (Bedoussac & Justes, 2010a; Lithourgidis et al.,
2011; Pelzer et al., 2012; Bedoussac et al., 2015), including
the reduction in numbers of cereal and legume aphids
(Ndzana et al., 2014; Lopes et al., 2015; Hatt et al., 2018;

© 2019 The Netherlands Entomological Society Entomologia Experimentalis et Applicata 168: 41–52, 2020 41
42 Mansion-Vaquie et al.
Xu et al., 2018). However, the ecological mechanisms at
the origin of these changes in abundance are still under
discussion.
Plant diversity may promote pest regulation either bot-
tom-up, driven by lower trophic levels, or top-down, dri-
ven by higher trophic levels (Gurr et al., 2017). Aphid
control seems to depend on both, with top-down effects of
natural enemies (specialists or generalists) being modu-
lated by interactions between aphids and their host plant
(Diehl et al., 2013). Several hypotheses explain how
increasing intrafield diversity may promote bottom-up
control. The ‘disruptive crop’ hypothesis (Vandermeer,
1989) suggests that the presence of a non-host plant would
lower a herbivore’s ability to locate and colonize its host
plant, because of chemical and physical confusion
imposed by a non-host species and/or because the herbi-
vore is more likely to leave a patch following repeated
encounters with a non-target plant. A non-host plant may
interfere with the chemical cues used by a herbivore to
locate its host plant by masking the host’s odours, altering
the host’s chemical profile, or introducing repellent com-
pounds (Randlkofer et al., 2010; Ben-Issa et al., 2017).
Concerning physical interference, Perrin & Phillips (1978)
refer to physical barriers, that is, the more arduous physi-
cal access of the herbivores to their host plants due to the
increased distance between host plants or the presence of
taller non-host plants. Additionally, camouflage in which
host plants might be hidden by the presence of other green
foliage or background (Smith, 1976; Francis, 1989) might
also create physical disturbance. A more complex architec-
ture of the vegetation would disrupt the herbivores’ beha-
viour and prevent them from easily reaching their host
plant. One of the most important ways in which aphids
find and select a host plant is by chemical cues (Webster,
2012; D€ oring, 2014), and chemical interference in aphid
host plant location by non-host plants has been demon-
strated (Xie et al., 2012; Ninkovic et al., 2013). However,
less attention has been paid to the role of physical interfer-
ence of non-host plants in the way aphids find their hosts.
Although aphids initially infest annual crops through
the migration of flying morphs from surrounding habitats
(Fievet et al., 2007; Irwin et al., 2007), later generations
mainly comprise wingless individuals. In this work we are
interested in this later phase of the infestation and on how
intercropping might disrupt the behaviour and population
growth of apterous aphids. Walking represents a frequent
mode of dispersal during an aphid’s lifetime (Irwin et al.,
2007): when the competition in a colony increases, local
dispersion, such as intra- or inter-plant movements,
acquires great importance and affects the aphids’ coloniza-
tion process (Lombaert et al., 2006). For instance, in an
alfalfa field experiment, the majority of apterous pea
aphids could walk up to 2 m within 4 h (Ben-Ari et al.,
2015). Although walking plays a decisive role in the spread
of the infestation at the field scale (Hodgson, 1991), on the
individual aphid scale, leaving a plant to look for another
is both risky and energy-expensive. Without a doubt, this
carries fitness costs, and thus trade-offs between foraging
and reproductive success are common (Stearns, 1992). In
aphids the various dispersal strategies between alate and
apterous individuals correspond to different sizes of
gonads, with alate aphids being less fecund than apterous
aphids (Dixon et al., 1993; Braendle et al., 2006). More-
over, apterous pea aphids that had dropped off or walked
away from their host plant to avoid predators were shown
to have reduced numbers of offspring, with consequences
for population growth, compared to apterous aphids
undisturbed by predators (Nelson et al., 2004).
In this study, we compared the host location behaviour
and population growth of apterous individuals of the cer-
eal aphid Sitobion avenae (Fabricius) in two intercropping
and one mono-cropping systems: soft winter wheat, Triti-
cum aestivum L. (Poaceae)–winter pea, Pisum sativum L.
(Fabaceae), soft winter wheat–white clover, Trifolium
repens L. (Fabaceae), and pure stands of soft winter wheat.
First, we hypothesized that aphids’ host plant location is
reduced in the presence of a non-host plant, and we con-
ducted a short-term behavioural experiment to evaluate it.
Second, in a long-term experiment we tested the hypothe-
sis that aphid population growth is limited in the presence
of a non-host plant. In this experiment, we also considered
that intercropping may affect plant quality, and in turn
may affect aphid performance, which is determined by
plant sap quality, especially in terms of nitrogen content
from free amino acids (Dinant et al., 2010). Finally, we
compared winter pea and white clover, two structurally
different non-host plants that could be used in wheat-
based intercropping systems (Lopes et al., 2016; Vrignon-
Brenas et al., 2018). Winter pea is characterized by an inde-
terminate growth (> 60 cm plant height) (Cousin, 1997;
Bedoussac & Justes, 2010b) and often produces tendrils
that enhance connectivity between plants, whereas white
clover mostly grows horizontally and stays low (20-30 cm
plant height) (Frame & Newbould, 1986; Frame, 2005).
Material and methods
Aphid stock cultures
A S. avenae aphid stock culture was initiated in the labora-
tory using individuals obtained from Koppert, Berkel en
Rodenrijs, The Netherlands (Ervibank). The colony was
maintained on winter wheat grown in 2-l pots in an
insect-free chamber controlled for light and temperature
(L16:D8, 21 °C).

Plants
Plants used in the experiments were grown from organic
seeds (wheat: Moulin Marion, St Jean sur Veyle; clover:
Jouffray Drillaud, Cisse; pea: Florimond Desprez, Cap-
pelle-en-Pevele, all in France), forming three experimental
treatments: wheat monoculture (W), wheat–winter pea
intercrop (WP), and wheat–white clover intercrop (WC)
(Figure 1). For all experiments, winter wheat (T. aestivum
cv. Renan) was grown in 4-l pots (18 cm diameter, 15 cm
high) containing Jiffy soil substrate (NFU 44-551) in an
insect-free chamber controlled for light and temperature
(L16:D8, 21 °C) and watered with 500 ml at seeding and
subsequently with 250 ml every 2 or 3 days. No fertilizer
was applied to any treatment because intercropping is
mainly used in low-N-input cropping systems and organic
farming, in particular for cereals (Bedoussac & Justes,
2010b). In a regular pattern, 36 wheat seeds were sown
per pot at 2 cm distance between seeds. Winter pea
(P. sativum cv. Enduro) was sown with wheat in an addi-
tive design by inserting one pea seed every three wheat
seeds for a total of 12 pea seeds per pot. White clover
(T. repens cv. Rivendel) was homogeneously sown
(70 mg, i.e., ca. 100 seeds per pot) in an additive design.
All the seeds were free of pesticide treatments. Seed densi-
ties correspond to the ones used in the field (Ndzana et al.,
2014; Vrignon-Brenas et al., 2018). In the few cases where
some seeds of wheat or pea did not germinate after 5–
6 days, they were replaced with plants grown under the
same conditions in extra pots. We counted 85 grown clo-
ver plants on average per experimental pot. Plants used in
the experiments were 7 days old.
Figure 1 Schematic representation of the experimental design: seeds of wheat, pea, and clover were sown in 4-l pots in the laboratory, to
form three treatments: wheat monoculture (W), wheat–clover intercrop (WC), or wheat–pea intercrop (WP).
Intercrops disrupt aphid behaviour and population 43
Behavioural experiment
In order to investigate the behaviour of apterous aphids in
the various treatments (W, WP, or WC), we ran an experi-
ment with five sessions, with 10 pots of each treatment per
session. Before starting, the vegetation cover was charac-
terized for each pot. We measured the height of five plants
of each species and the number of contacts among plants
(i.e., among wheat plants and between wheat and associ-
ated plants). Additionally, we calculated the percentage of
total ground area covered by vegetation: each experimental
pot was photographed from above and the photographs
were later analysed using ImageJ v.1.44o software
(National Institute of Health, Bethesda, MD, USA). The
experiment took place in an insect-free chamber con-
trolled for light and temperature (L16:D8, 21 °C).
Experimental aphids were standardized for age prior to
the experiments; adult aphids were individually placed in a
glass tube (2 cm diameter, 15–20 cm high) on a 7-day-old
wheat plant, previously transplanted from soil to a wet cot-
ton wool substrate. After 48 h, the adult aphid was
removed and the newborn nymphs were kept in the tube
for 7–8 days until they became adults. They were then
transferred to an insect-free chamber controlled for light
and temperature (L16:D8, 21 °C), where the experiment
took place.
Aphids were starved for 3 h before the beginning of the
experiment to reduce the variability due to individual
physiological conditions (Quiroz et al., 1992; Caillaud &
Via, 2000). Then, using a fine brush, each aphid was care-
fully placed on the ground at the centre of a pot, and once
the aphids started to move, the time spent on the wheat or
44 Mansion-Vaquie et al.
the associated non-host plant or the ground was recorded
continuously for 5 min. Additionally, the position of the
aphid was recorded at 10, 20, 30, and 60 min after the
beginning of the experiment. Due to the small size of the
clover, it was difficult to follow the movement of the aphid
from one individual clover plant to another without dis-
turbing it; therefore we considered visits to the clover
cover as a whole and recorded when it moved from the
clover cover to the ground, or from clover to wheat plants
and vice versa. Whenever the aphid reached the edge of
the pot, the observation stopped and the aphid was con-
sidered to be escaping and so was removed from the exper-
iment. In total 149 aphids were monitored.
Aphid population growth experiment
In order to investigate the aphid population growth in the
various treatments, we used 66 experimental pots per
treatment (i.e., 198 experimental pots in total). On the first
day of the experiment and for each treatment, half of the
experimental pots were inoculated each with 10 apterous
aphids: five standardized adults (same protocol as above)
and five randomly selected last instars from the stock cul-
tures; the remaining pots were the non-infested controls.
All pots were enclosed in a plastic frame (37 cm high,
20.5 cm diameter) covered with a tight meshed net, which
prevented the aphids from either escaping from the
infested pots or entering the control pots. Then, 7, 14, and
21 days after inoculation, 22 pots per treatment (11
infested and 11 control) were randomly removed from the
experiment. In each infested pot, aphids were manually
collected from the plant and placed in a Petri dish (55 mm
diameter), which was then kept at 80 °C. Later, the
winged and wingless adults and nymphs were counted
under a stereo microscope.
The wheat above-ground biomass was collected, dried
at 60 °C for 48 h, and weighed using a portable laboratory
Table 1 Statistical analysis for each variable in the behavioural experiment and the explanatory fixed factors and random factors, if
applicable
Variables Statistical test/model
% aphids leaving the first plant Binomial GLMM
encountered
No. visits of plants or ground Poisson GLMM
No. escaped aphids Poisson GLMM
No. contacts among plants Poisson GLMM
Time spent on plants LMM
Vegetation cover LMM
Wheat height LMM
Time needed to reach the first wheat plant Log-rank test
Probability to encounter wheat first Exact binomial test
NA, not applicable.
scale PT210-000V1 balance (Sartorius, G€ ottingen, Ger-
many), except 2.5 g of the fresh wheat biomass, that was
kept aside at 20 °C for chemical analyses in order to
measure the concentration of free amino acids in the
wheat leaves. These chemical analyses were performed as
follows: the wheat leaves were dried in a vacuum oven
(40 °C and 1 bar) for 36 h and then shredded with a ball
mill; 20 mg of this wheat leaf powder was diluted in
500 ml distilled water and heated at 70 °C for 1 h. After
cooling, the solution was centrifuged for 5 min at
10 000 rpm and the resulting supernatant was analysed
with the K-PANOPA kit (Megazymes, Bray, Ireland), in
accordance with the manufacturer’s protocol.
Statistical analysis
Data of the behavioural experiment were analysed using
mixed models (Table 1), with ‘experimental session’ as a
random factor to take into account the dependency of
observations of experimental pots made on the same date.
Generalized linear mixed models (GLMMs) and linear
mixed models (LMMs) were built with the glmer and lmer
functions, respectively, from the lme4 package (Bates
et al., 2015).
The significance of fixed factors and their interaction
was determined by an F-test with a Kenward-Roger cor-
rection for LMMs or likelihood ration tests (LRT) for
GLMMs, as implemented in the mixed function in the afex
package (Singmann et al., 2018). If non-significant, the
interaction was removed from the model and the signifi-
cance of fixed factors was re-tested. The time that the
aphids needed to reach their first wheat plant was com-
pared among treatments with a survival time analysis
using the log-rank test from the survfit function in the sur-
vival package (Therneau & Lumley, 2017).
Aphid numbers in the population growth experiment
were compared with a quasiPoisson GLM to account for
Fixed factors Random factors
Treatment, identity of first plant Experimental session
encountered
Treatment Experimental session
Treatment Experimental session
Treatment Experimental session
Treatment Experimental session
Treatment Experimental session
Treatment Experimental session
Treatment NA
NA NA
 Intercrops disrupt aphid behaviour and population 45

Table 2 Statistical analysis for each variable in the aphid popula- overdispersion (Table 2). When non-significant, the inter-
tion growth experiment and the explanatory fixed factors action was removed from the model and the significance
of fixed factors was re-tested. Aphid densities (number of
Variables Statistical test/model Fixed factors aphids over plant weight) were analysed with a non-linear
Aphid abundance quasiPoisson GLM Treatment, days quadratic regression. The demographic composition of
Aphid densities Quadratic (non- Treatment, days the aphid populations at single dates was analysed with
linear) regression ANOVA, or with Kruskal–Wallis rank sum test if data
Adult aphid ANOVA Treatment were not normally distributed. For the ANOVA, all possi-
densities ble interactions were considered and non-significant inter-
Winged aphid Kruskal–Wallis rank Treatment actions were removed using a stepwise model selection by
densities sum test
Akaike’s information criterion (AIC). For all models and
Wheat dry biomass ANOVA Treatment, aphid
when appropriate, pairwise comparisons were realized
(presence or
absence), days using Tukey-adjusted least-square means (LSMeans) with
Free amino acid ANOVA Treatment, days the emmeans package (Lenth, 2018). All statistical analyses
concentration were conducted in R v.3.4.3 software (R Core Team 2018).

Results
Table 3 Aphid behaviour in each of three treatments [wheat
Behavioural experiment
monoculture (W), wheat intercropped with pea (WP), and wheat
intercropped with clover (WC)]: the first plant encountered Due to the difference in seeding densities, the probability
(wheat host or non-host) and whether the aphids leave (i.e., walk that an aphid by chance first encountered a wheat plant
away from the first plant encountered) or stay for the duration of was 0.75 for WP, 0.30 for WC, and obviously 1 for W. Our
the assay (5 min) observations did not differ from this (Table 3): 66% of
aphids first visited a wheat plant in WP (Exact binomial
Treatment test: 95% confidence interval = 0.51–0.79, P = 0.14),
WP whereas only 30% of aphids first visited a wheat plant in
W (n = 49) (n = 50) WC (n = 50) WC (Exact binomial test: 95% CI = 0.18–0.45, P = 1.0).
All aphids reached a wheat plant in W. The time needed
First plant visited
for aphids to reach a wheat plant differed among treat-
Identity Wheat Wheat Pea Wheat Clover None
Rate (%) 100 66 34 28 70 2
ments (log-rank test: v2 = 17.9, d.f. = 2, P<0.001): it was
Behaviour longer in WC (70 s) than in WP (33 s) and W (27 s)
Leave (%) 57 34 26 16 56 – (Figure 2).
Stay (%) 43 32 8 12 14 – All 149 aphids except one visited at least one plant dur-
ing the first 5 min of the experiment, but more than 50%

Figure 2 Kaplan–Meier plot of the time

needed for aphids to reach a wheat plant
during the first 5 min in each of three
treatments: wheat monoculture (W),
wheat intercropped with clover (WC), and
wheat intercropped with pea (WP).
46 Mansion-Vaquie et al.
of the aphids left the first plant that they visited during this
period (Table 3). The probability of leaving the first plant
visited was greater when the plant was a non-host plant
(LRT on GLMM: v2 = 5.60, d.f. = 1, P = 0.02) and this
probability was similar across treatments (v2 = 0.37,
d.f. = 2, P = 0.83).
During the first 5 min, aphids came back to the
ground more often in WC (1.39 on average) compared
to the other treatments with less than one return on aver-
age (LRT on GLMM: v2 = 14.69, d.f. = 2, P<0.001).
Aphids visited a similar number of wheat plants (1.5 on
average) whatever the treatment (v2 = 3.43, d.f. = 3,
P = 0.33), but spent more time on wheat in the W
(234 s) and WP (195 s) treatments than in the WC
(139 s) treatment (F-test on LMM: F2,140 = 14.5,
P<0.001) (Figure 3). Concerning the non-host plants,
aphids spent more time on clover (97 s) than on pea
(49 s) (F-test on LMM: F1,92 = 6.9, P = 0.01).
During the 1-h experiment, 27% aphids left the experi-
mental pot; the rate of escape was not different among the
Figure 3 Mean (+ SEM) cumulated residence time (s) of aphids
on wheat host plants and on clover or pea non-host plants during
the first 5 min in each of three treatments: wheat monoculture
(W), wheat intercropped with clover (WC), and wheat
intercropped with pea (WP). Means within a plant type capped
with different letters are significantly different (Tukey-adjusted
pairwise LSMeans comparisons: P<0.05).
treatments (LRT on GLMM: v2 = 0.12, d.f. = 2,
P = 0.94). Of the remaining aphids, 94 and 88% visited at
least one wheat plant in the WP and WC treatments,
respectively (v2 = 0.49, d.f. = 1, P = 0.49).
Differences in aphid behaviour among treatments may
be related to contrasting vegetation cover (Table 4). As
expected from seeding densities, ground cover differed
among treatments (F-test on LMM: F1,149 = 17.65,
P<0.001) – it was largest for WC, intermediate for WP,
and lowest for wheat in monoculture. Pea plants never
touched each other, whereas clover plants formed a con-
tinuous cover such that it was difficult to distinguish every
single contact among plants. Wheat plants were signifi-
cantly more in contact with clover than with pea plants
(Table 4).
Aphid population growth experiment
Aphid populations increased over the 3-week experiment
(Figure 4) (GLM: F1,91 = 223.97, P<0.001) and aphid
number differed among treatments (F2,92 = 5.68, P<0.01)
– intercropping wheat with a legume reduced the number
of aphids compared to solely wheat, but no difference
between WC and WP was observed (Tukey-adjusted pair-
wise LSMeans comparisons: P<0.05). We did observe,
however, differences among wheat biomasses from differ-
ent treatments at 21 days of infestation (Figure 5) – the
biomass of wheat associated with pea was significantly
lower when compared to wheat in monoculture and to
wheat associated with clover. Although there were
differences among treatments, the biomasses of wheat
Table 4 Vegetation characteristics: mean ( SEM) ground cover
(%), number of contacts between wheat plants and between
wheat and non-host plants (clover and pea), and plant height
(cm) in each of three treatments: wheat monoculture (W), wheat
associated with clover (WC), and wheat associated with pea
Treatments
W WC WP
Ground cover (%) 13.4  0.4a 16.5  0.4b 14.8  0.4c
No. contacts among 6.0  0.5a 6.8  0.4a 4.4  0.4b
wheat plants
No. contacts – 6.9  0.4a 2.3  0.2b
between wheat and
non-host plants
Wheat height (cm) 13.4  0.2a 13.6  0.2a 12.7  0.2b
Non-host plant – 1.4  0.0 4.3  0.1
height (cm)
Means within a row followed by different letters are significantly
different (Tukey-adjusted pairwise LSMeans comparisons:
P<0.05; applied on LMMs for vegetation cover and wheat height,
or GLMMs for contacts among plants).
 Intercrops disrupt aphid behaviour and population 47

Figure 4 Mean ( SEM) number of aphids

over 3 weeks after inoculation in each of
three treatments: wheat monoculture (W),
wheat intercropped with clover (WC) and
wheat intercropped with pea (WP).
Dashed lines result from the quasiPoisson
GLM analysis that best described the data.

infested with aphids were significantly lower compared to
the control plants. Therefore, taking into account aphid
densities – number of aphids per g of dry wheat mass (Fig-
ure 6) – intercropping wheat with clover reduced aphid
densities compared to sole wheat and to wheat inter-
cropped with pea (Tukey-adjusted pairwise LSMeans
comparisons: P<0.05).
The demographic composition of the aphid populations
indicated that aphid populations were dominated by
apterous nymphs (Table 5). The density (number of
aphids over wheat biomass) of winged adults and nymphs
was negligible at the first two sampling dates and still rep-
resented less than 2% of the aphid population at 21 days.
At this last sampling date, the mean density of winged
aphids was not significantly different between treatments
(Kruskal–Wallis rank sum test: v2 = 0.66, d.f. = 2,
P = 0.72). However, it should be noted that winged aphids
were present in the WC treatment since the first sampling
date, contrary to the other treatments where they only
appeared in the 21-days sampling.
In order to explore a possible relationship between
aphid population growth and plant sap quality when inter-
cropped, we analysed the concentration of free amino
acids in leaves of the non-infested wheat plants (i.e., con-
trols) of the various treatments (Figure 7). Free amino
acids decreased over time (ANOVA: F2,87 = 63.07,
P<0.001) and their concentration was not different among
treatments (F2,87 = 1.60, P = 0.57). There was a signifi-
cant interaction between time and treatment (F4,87 = 7.56,
P = 0.04) due to the significant difference in free amino
acid concentration in wheat plants from W and WP treat-
ments 14 days after aphid infestation, but this difference
disappeared later on.
Discussion
This laboratory study aimed at testing the hypotheses that
combining wheat and legumes modifies the behaviour of
the cereal aphid S. avenae in terms of host plant location
and population growth. The aphids’ residence time on

Figure 5 Mean ( SEM) wheat dry

biomass (g) at 1, 2, and 3 weeks after
aphid inoculation, in the presence or
absence (control) of aphids, in each of
three treatments: wheat monoculture (W),
wheat intercropped with clover (WC), and
wheat intercropped with pea (WP). Means
capped with different letters are
significantly different (Tukey-adjusted
pairwise LSMeans comparisons: P<0.05).
48 Mansion-Vaquie et al.

Figure 6 Mean ( SEM) aphid densities

(no. aphids per g wheat dry biomass) over
3 weeks after aphid inoculation in each of
three treatments: wheat monoculture (W),
wheat intercropped with clover (WC), and
wheat intercropped with pea (WP).
Dashed lines result from the quadratic
regression analysis that best described the
data.

wheat decreased when this host plant was intercropped biomass, only intercropping clover with wheat signifi-
with clover. At the population level, wheat–legume inter- cantly reduced aphid densities on wheat – intercropping
crops reduced the number of aphids on wheat plants com- clover with wheat was particularly disruptive to S. avenae
pared to sole crops of wheat, but if we account for plant behaviour.
Our observations support the first hypothesis that host
location by apterous aphids is impeded by the presence of
Table 5 Mean ( SEM) densities (number of aphids per g plant) a non-host plant, corresponding in our experiment to an
of apterous and winged aphids according to their growth stage additive design simulating field seed densities of wheat–
and the treatments: wheat monoculture (W), wheat associated
pea and wheat–clover intercrops. Although connectivity
with clover (WC), and wheat associated with pea (WP), at 7, 14,
between plants was higher in the wheat–clover treatment,
and 21 days after infestation
this did not help apterous aphids to more easily reach their
Treatment host plant. Chemical and physical interference might
explain this result, as they are both involved in host loca-
W WC WP
tion (Gish & Inbar, 2006; Reeves, 2011; Webster, 2012).
7 days Both alate and apterous aphids preferred the volatile
Apterous organic compounds emitted by pure stands of wheat com-
Adults 2.0  0.5 1.2  0.5 1.4  0.4 pared to those emitted by intercrops (Xie et al., 2012; Nin-
Nymphs 57.4  12.3 41.8  6.3 43.3  5.7 kovic et al., 2013). Odours therefore play a role as first
Winged attractors to host plants. However, the rejection of a non-
Adults 0 0.3  0.2 0
host plant by insects occurs after reaching the plants, not
Nymphs 0 0 0
before (Finch & Collier, 2012). Once on the plant, aphids
14 days
Apterous initiate a second recognition process with their antennae
Adults 7.3  1.3 5.1  0.9 9.8  1.3 by probing the plant to obtain physical and chemical
Nymphs 43.3  5.6 31.6  6.7 41.3  6.1 information about its suitability (Caillaud & Via, 2000).
Winged This recognition stage takes around 60 s and is essential to
Adults 0 0.1  0.1 0 avoid full stylet penetration on the wrong plant, which is
Nymphs 0 0 0 time-consuming (45 min; Dixon 1998). Therefore, in the
21 days case of high non-host plant density, this second stage may
Apterous have to be repeated for each encountered plant, increasing
Adults 14.0  1.8 10.8  1.6 12.1  2.2 the time necessary to ultimately come upon the host plant.
Nymphs 90.7  10.9 61.1  8.3 79.1  12.2
Indeed, in our experiment we observed that intercropping
Winged
wheat with clover increased the aphids’ movements
Adults 0.3  0.1 0.4  0.3 0.1  0.1
Nymphs 1.2  0.5 0.8  0.3 0.9  0.4 between the ground and the clover cover and within the
clover cover, significantly delaying the aphids’ arrival to

Figure 7 Mean ( SEM) free amino acid
content (mg g 1) in leaves of non-infested
wheat plants after 1, 2, and 3 weeks in each
of three treatments: wheat monoculture
(W), wheat intercropped with clover
(WC), and wheat intercropped with pea
(WP). Means capped with different letters
are significantly different (Tukey-adjusted
pairwise LSMeans comparisons: P<0.05).
the wheat. Although our experiments were not designed to
test for the effect of plant density, these observations point
out that the ratio of the two intercropped plants may be
important for pest control.
Concerning the second hypothesis, the aphid popula-
tion growth suggests bottom-up regulation through plant
interspecific diversification. However, intercropping can
reduce intercropped plant mass because of interspecific
competition (Thorsted et al., 2006) and generate con-
founding effects that complicate the evaluation of herbi-
vore responses to intercropping (Bukovinszky et al., 2004).
Therefore, host plant biomass should be taken into
account when considering intercrop infestation effects
(Ndzana et al., 2014). This means that S. avenae infestation
was only significantly lower for the wheat–clover intercrop
treatment.
The lower number of aphids in wheat–clover intercrops
may be explained by slower female development,
decreased fecundity, increased mortality, and/or a higher
level of emigration. Emigration can be discarded in our
experimental setup because each pot was isolated under a
net. However, in the wheat–clover treatment alate aphids
appeared since the first week of infestation, indicating that
migration was induced and thus could have strengthened
the decrease in aphid numbers if winged aphids had been
allowed to leave the experiment.
All these processes could be influenced indirectly by the
effect of cover structure on aphids’ behaviour and/or
directly by plant quality. On the one hand, the increased
walking activity in wheat–clover treatments, observed in
the first experiment, could carry fitness costs which are
known to trade-off with reproductive success (Stearns,
1992), with consequences for population growth (Hooks
& Fereres, 2006). Moreover, repetitive probing of a non-
Intercrops disrupt aphid behaviour and population 49
host plant associated with failure in host location is known
to induce flight in aphids (Finch & Collier, 2000). On the
other hand, cereals and legumes compete for soil nutrients
at early growth stages (Corre-Hellou et al., 2006; Bedous-
sac et al., 2015). Such competition may impact plant qual-
ity and constrain the growth rate of aphids (Theunissen,
1994; Dixon, 1998) and induce the production of winged
aphids to escape unsuitable conditions (Braendle et al.,
2006; Awmack & Leather, 2007; Irwin et al., 2007). Never-
theless, in our experiment, we did not find a decrease in
the biomass, nor differences in the concentration of free
amino acids in the leaves of wheat intercropped with clo-
ver, compared to that from wheat monocultures. Unfortu-
nately, our method does not distinguish essential from
non-essential amino acids which, together with the ongo-
ing debate about the pathways of uptake and utilization of
plant nutrients by aphids, muddles the link between sap
quality and aphid population growth (Vogel & Moran,
2011; Haribal & Jander, 2015).
Finally, the standardized design of our laboratory exper-
iments assumed an equal inoculum of apterous aphids
across treatments, but as intercropping might reduce the
initial colonization of the crop by winged aphids (Finch &
Collier, 2000; D€ oring, 2014), aphid population growth
may differ under field conditions. Moreover, our results
only concern the first 4 weeks of plant growth, whereas
S. avenae mainly colonizes later wheat developmental
stages.
Flight arrival of aphids on a crop has been shown to
decrease with increased vegetation cover and diversity
(Finch & Collier, 2000; D€ oring, 2014). Although it has
been argued that increasing vegetation diversity may be
more effective in reducing the winged aphid colonizers of
wheat than in regulating the later spread of aphid colonies
50 Mansion-Vaquie et al.
(Costello & Altieri, 1995; Wratten et al., 2007), our study
shows that vegetation diversity can also contribute to
decreasing the growth of the later wingless aphid popula-
tions. Nevertheless, this depends on the species used as
non-host plants and their density. Although additive vs.
substitutive designs are often compared in studies of the
effect of intercropping on pest control, little is reported
about the influence of plant relative densities on pest dis-
persal (Lopes et al., 2016). Our study points out that these
are important parameters that should be taken into
account. In our experimental set up that copied additive
designs used in real-farming conditions, intercropping
clover with wheat was particularly disruptive to apterous
S. avenae and may be an interesting strategy for its control.
Nevertheless, our observations are valid only for aphid col-
onization of early plant growth stages. Later on, the vegeta-
tion structure as well as interspecific interactions –
competition for nutrients – may change and influence
aphid behaviour differently; this should be further investi-
gated. Finally, our laboratory results need to be confirmed
at a larger scale under actual field crop conditions, where
other parameters such as the interaction with natural ene-
mies might play an important part in the final aphid con-
trol (Vidal & Murphy, 2018).
Acknowledgements
The authors would like to thank N Dardenne, G Espinasse,
and J-F Garrigues for technical assistance, and V Tolon for
support with statistical analyses. We are also grateful to A
Sentis and J-L Hemptinne for general advice on the experi-
mental set and we acknowledge the critical reading of the
manuscript by J-L Hemptinne. This work was funded by
the Compagnie Nationale du Rh^ one (CNR), France. Pea
seeds were graciously offered by Florimond-Desprez. The
laboratory ‘Evolution et Diversit
e Biologique’ is part of the
‘Laboratoire d’Excellence’ LABEX TULIP (ANR-10-
LABX-41).
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Experimental set up of the behavioural
experiment: (A) overall configuration and an experimental
pot of the treatment: (B) wheat monoculture, (C) wheat
intercropped with pea, and (D) wheat intercropped with
clover.